The overall goal of the K08 award is to permit Laura J. Rush, D.V.M., to devote full-time effort for research training leading to a Ph.D. degree in molecular biology, and for development into an independent scientist for a career in academic biomedical research. Dr. Rush has completed a residency in veterinary and comparative pathology and 3 years of research in the laboratories of Michael A. Caligiuri, M.D., and Christoph Plass, Ph.D., co-sponsors of this award. Acute myeloid leukemia (AML) is a heterogeneous disease and the pathogenesis of most types of AML is unknown. The overall hypothesis of this research project is that aberrant DNA methylation in AML is associated with inactivation of genes that are necessary for normal growth, differentiation and/or death of hematopoietic cells, and t h at these epigenetic alterations play a key role in the molecular pathogenesis of AML. The proposed project will focus on fifteen candidate genes that have been identified by genome scanning experiments performed by Dr. Rush using primary AML samples. The goals of the project are to use these 15 genes to: 1) determine the consequences of restoration of gene expression on morphology, differentiation, and growth characteristics in vitro using AML cell lines; 2) investigate the methylation status of the 5' CpG islands by molecular techniques such as bisulfite treatment and methylation-specific in situ hybridization; and 3) correlate aberrant promoter methylation with transcriptional inactivation in AML diagnostic samples. Genes will be prioritized based on expression in normal hematopoietic tissues. The genes will then be transfected into non-expressing AML cell lines to elucidate what effects restoration of expression may have on growth rate, differentiation, b l a s t - colony forming ability, and morphology (Aim 1). Extensive characterization of the methylated 5' regulatory regions of these genes will be performed and results correlated with the presence or absence of the protein product (Aim 2). Dr. Rush has already developed extensive preliminary data for this project. The cooperative efforts of Dr. Rush, the co-sponsors, and co-investigators in the Division of Human Cancer Genetics, Comprehensive Cancer Center, and the Cancer and Leukemia Group B Tissue Bank will provide a productive environment to complete this significant investigation. The combination of results from studies of primary tumors, in vitro cultures, and mechanistic molecular experiments will contribute to the understanding of the role of aberrant DNA methylation in leukemogenesis.